The subject invention relates an anti-fungal preparation for the control of fungal diseases in plants, more specifically, to alkaline extracts of Inula which are highly active in controlling diseases caused by fungi in crop plants.
Extracts of plants which are members of the Inula species are effective against infections of plants caused by a variety of fungi. These extracts are typically prepared by dipping freshly cut Inula shoots in an organic solvent or by agitating freshly cut or dried Inula shoots in an organic solvent, removing the solvent to form a paste, and then dissolving the paste in an organic solvent or in water, possibly with an additive. Alternatively, suspensions of Inula can be prepared by grinding dried Inula shoots into a fine powder, adding an emulsifier, and then suspending the mixture in water. In both cases, the resultant preparation is then applied to plants resulting in the control of a wide variety of fungal diseases. Extracts of suspensions of Inula plants are effective at low concentrations, in the range of fractions of a single percent of extract, such that dilute concentrations have excellent fungal-control properties.
The plants which are the basis of these fungicidal extracts and suspensions are Inula viscosa and Inula graveolens (Family Compositae), perennial weeds widespread in the Mediterranean Basin.
Methods of preparing aqueous extracts from various parts of the Inula plant are well known in the literature. Additionally, organic extracts of the Inula plant are also well known in the art, e.g., U.S. Pat. No. 5,837,253. However, the prior art indicates that aqueous extracts of Inula shoots are poorly effective against fungal diseases of crop plants whereas extracts made with organic solvents are highly effective anti-fungal agents.
Methods have also been described for using organic solvents to extract Inula plants, but these are clearly distinguished from that disclosed in U.S. Pat. No. 5,837,253. Two of the prior art methods involved contacting the whole Inula plant, or the aerial parts thereof, with an organic solvent either by maceration of the plant in the organic solvent or by percolation of the solvent through the plant. Furthermore, U.S. Pat. No. 4,254,112 to Debat et al., (hereinafter referred to as xe2x80x9cDebatxe2x80x9d) describes the preparation of extracts of Inula viscosa and Inula graveolens using whole Inula plants which have been dried and ground and organic solvents, by using a Soxhlet apparatus. The yield of the paste obtained by this method was approximately 1.75-4%. U.S. Pat. No. 5,176,193 to Honerlagen et al. (hereinafter referred to as xe2x80x9cHonerlagenxe2x80x9d) describes a process for preparing a partial extract from roots of Inula helenium which involves contacting the plant material with an organic solvent, adding a drying agent to the solution to remove the water, removing the drying agent, and then distilling the dried organic phase. By contrast, the method disclosed in U.S. Pat. No. 5,837,753 disclosed either briefly dipping the leaves and stems of the shoots of Inula viscosa or Inula graveolens into an organic solvent or shaking the freshly cut or dried and ground leaves and stems of the shoots in an organic solvent for thirty minutes, and then evaporating the solvent to form a paste. The yield obtained by this method can be as much as 30%, in contrast to the low yields known in the literature and described above.
The medicinal properties of Inula extracts in humans are well known. For example, Debat disclosed the anti-microbial activity of extracts of Inula for use in human beings. However, the fungicidal effects of Inula extracts have only been demonstrated on fungi growing in Petri dishes or on post-harvest of fruits. For example, Qasem et al. (Phytopathologia Mediterana, 34:7-14, 1995) demonstrated that the growth of certain fungi in Petri dishes was inhibited by aqueous extracts of Inula viscosa as well as by dried plant material added directly to the fungal growth media. By contrast, the method of the present invention uses Inula extracts prepared with alkaline aqueous solvents used against fungal infections of crop plants themselves.
Clearly, although Inula extracts have been shown to have fungicidal activity in the Petri dish (in vitro) and on plant (in vivo), the methods of preparation for these extracts have not been sufficient for large scale use directly on crop plants and have had other serious disadvantages. The true effectiveness of these extracts against fungal infections of plants is, therefore, unknown. Furthermore, there is a clear need for better methods to prepare Inula extracts. Qasem et al. (Ibid, page 13, 1995) concluded: xe2x80x9cThe diversity and the methodology of extraction and the differences in the results obtained . . . increased the need for developing more efficient, convenient, and cheaper methods of extraction to facilitate more extensive utilization of fungicidal extracts, especially if greater quantity of extracts must be prepared for large-scale productionxe2x80x9d.
Thus, there is a widely recognized need for and would be highly advantageous to have a method or methods for preparing extracts and/or suspensions of plants of Inula species which would facilitate the large-scale use of these extracts and suspensions which would simplify their preparation and use, be highly effective in controlling fungal infection in plants, as well as controlling fungal infections in crop plants.
According to the present invention, there is provided a method for preparing an aqueous extract from Inula species which comprises contacting shoots and/or portions thereof with an alkaline aqueous solvent to form a solution and debris, and removing the debris from the solution.
There is also provided a method for protecting plants against fungal infection comprising preparing a fungicidal extract of Inula species by contacting shoots and/or portions thereof with an alkaline aqueous solvent to form an extract solution and debris, and applying a fungicidally effective amount of the fungicidal extract to a plant for protecting the plant against fungal infection.
There is also provided in accordance with the present invention, a method for preparing a fungicide derived from Inula species which comprises combining a substantially powdered form of Inula shoots and/or portions thereof with a solid chemical to form a mixture which upon dissolution of the mixture in an aqueous solvent, forms an alkaline aqueous solution which can be directly applied to plants to prevent fungal infection.
Additionally, also in accordance with the present invention, there is provided an alkaline anti-fungal composition which comprises Inula shoots and/or portions thereof and a compound which when mixed with an aqueous solvent forms an alkaline solution, whereby the composition can be directly applied to plants to prevent fungal infection.
Accordingly, the subject invention provides a method for unexpectedly producing an anti-microbial extract from Inula plants which is not only active in vitro against fungi but also is active against diseases caused by fungi in crop plants while being safe to apply to the crop itself.
The present invention provides a method for preparing an extract from Inula species including the steps of contacting shoots and/or portions thereof with an alkaline aqueous solvent to form a solution and debris and then removing the debris from the solution.
Preferably, the portion and/or portions of the Inula plant which are utilized in the present invention are the leaves and stem of the upper, younger parts of the shoots, and the upper part of the shoot extending from about 20-40 centimeters from the tip of the shoot. The flowers themselves are not used. As used herein, the term xe2x80x9cshootxe2x80x9d is used to define the leaves and stem of the shoot of the Inula plant. In the methods of the present invention, the shoots or portions thereof can be either freshly cut or may be dried prior to the addition of the alkaline aqueous solvent.
The alkaline aqueous solvent can include any compound or compounds which alone or in solution have a pH which is greater than about seven such as an inorganic base or salt thereof.
Preferably, the alkaline aqueous solvent includes at least one of amino compound. The amino compound can include an ammonium compound, organic amines and/or heterocyclic amines. The organic amines can include compounds such as ethylamine, ethylenediamine, diaminopropane, diethanolamine, triethanolamine, and butylamnine. Heterocyclic compounds can include compounds such as piperazine and piperidine.
The removal of the debris from the solution can be accomplished by filtering the debris from the solution utilizing a screen such as a 100 micron xcexcm (micron) sieve or other suitable filtering devices known to those skilled in the art.
If dried shoots and/or portions thereof are utilized in the method of the present invention, the dried shoots and/or portions thereof can be ground, by various means known to those skilled in the art, to form a powder.
In an embodiment of the invention, fresh Inula shoots are dipped in the alkaline solution for an appropriate period of time, typically for approximately 0.2-5 minutes. The plant material is then discarded and the solution, which may be colored, obtained therefrom is screened through a filter such as a 100 micron sieve and can be applied, as is, for spraying crop plants.
In another embodiment, fresh shoots are first air dried at 30-40xc2x0 C. for period of time sufficient to dry the shoots, typically approximately 1-2 days and the dried shoots then dipped in the alkaline solution for a period of time which can be approximately 1-10 minutes. The plant material is discarded and the solution (colored) obtained can be filtered through a filter or sieve and then utilized, as is, for spraying crop plants.
In another embodiment, dried shoots obtained by air drying as described immediately above can be ground and then passed through a metal sieve (i.e., 2 mm). The ground plant material can then be shaken in an alkaline solution for 1-10 minutes, screened through a filtering device such as a 100 micron sieve, and the colored liquid obtained therefrom can be utilized for spraying crop plants.
In yet another embodiment, dried and ground plant material as immediately described above, can be placed in a mortar and is milled to less than 100 micron-sized particles to obtain a fine powder. The powder is then mixed with an alkaline solution and can be sprayed, as such, onto the crop plants.
In another embodiment, dried powder which is ground to less than 100 micron-sized particles is milled with crystallized cellulose (Avicel PH-101, Fluka) and is then mixed with the alkaline solution and sprayed directly onto crop plants.
In another embodiment, a dried powder which has been milled to less than 100 micron-sized particles is milled with a surfactant such as Vercoryl-S, diatomaceous earth, or sodium dodecyl sulfate and is then mixed with the alkaline solution and is sprayed directly onto the crop plants.
In another embodiment, dried Inula shoots ground to less then 100 micron-sized particles are mixed with a solid chemical, which upon dissolution in water, produces an alkaline solution. The solid chemical can be any of the compounds described above for the alkaline solution including an imidazole, dibasic ammonium phosphate, Trizma-base (Sigma) either with or without the addition of an inert surfactant formulation. The powder mixture is then mixed with water to form an anti-fungal agent and is sprayed directly onto crop plants. In this form, the anti-fungal agent can be prepared and/or sold as a dry mix which, when hydrated, forms an anti-fungal composition which can be easily applied to plants. In this form, the costs of shipping and/or transporting the anti-fungal material can be reduced since the water would only be added just prior to use/application to plants.
In another embodiment, the dried and ground Inula of particle size less than 100 microns can be mixed with (NH4)2HPO4 (dibasic ammonium phosphate) and be suspended in water to which NH4OH is added. This solution can then be sprayed directly onto crop plants.
According to the present invention, there is also provided a method for protecting plants against fungal infections by the steps of (a) preparing a fungicidal extract of Inula species by contacting shoots and/or portions thereof with an alkaline aqueous solvent to form an extract solution and debris and (b) applying a fungicidally effective amount of the fungicidal extract to a plant for protecting a plant against fungal infection. As defined herein, a fungicidally effective amount includes such an amount that is capable of preventing, inhibiting, and/or killing fungus in or on a plant.
Solutions or emulsions containing the Inula extract, or the Inula suspension, are sprayed onto the upper leaf surfaces of various crop plants by well known techniques for the control of fungal plant infections. The Inula extracts of the present invention can be used to control fungal plant infections caused by fungi including, but not limited to, Oomycetes, Ascomycetes, Basidiomycetes, and Fungi imperfecti classes.
The method and fungicidal extracts of the present invention are particularly suitable for use against fungi of the Oomycetes, Ascomycetes, Basidiomycetes and Fungi imperfecti classes including but not limited to Phytophthora infestans, Pseudoperonospora cubensis, Plasmopara viticola, Sphaerotheca fuliginea, Cladosporium cucumerinum, Erysiphe graminis, Uromyces appendiculatus, and Botrytis cinera and in crops including, but not limited to, grapevines, tomato, wheat, barley, tobacco, potatoes, onions, cucurbits, beans and crucifers.
Also according to the present invention, there is provided a method for preparing a fungicide derived from Inula species which includes combining a substantially powdered form of Inula shoots and/or portions thereof with a solid chemical to form a mixture which upon dissolution of the mixture and an aqueous solution, forms an alkaline aqueous solution which can be directly applied to plants to prevent fungal infection. Fungicides prepared by this method are described above and in the Example section below.
Also in accordance with the present invention, an alkaline anti-fungal composition of Inula shoots and/or portions thereof and a compound which when mixed with an aqueous solvent forms an alkaline solution which can be directly applied to plants to prevent fungal infection is disclosed. The compounds which can be mixed with an aqueous solution to form an alkaline solution are described above and in the Examples section below.
The invention of the present invention is illustrated below in the following Examples which describe the preparation and use of Inula extracts and suspensions against fungal infections in plants.
To test the efficacy of these extracts and suspensions, experiments were performed in which plants were sprayed using a fine glass atomizer with either Inula extract or with pure solvent as a control for the Inula extracts, with either Inula extract and emulsifier or with an emulsifier solution as a control for the emulsified Inula extracts, and with either Inula suspension or with water or emulsifier solution as a control for the Inula suspensions. Treated and control plants were then inoculated with a crop-respective fungal pathogen. After an incubation period, the extent of the infection was measured. Unless otherwise stated, percentage protection from the disease due to the treatment with the Inula extract or suspension was calculated as:
% control of the disease=[1xe2x88x92(% infection and treated plants/% infection in control plants)]xc3x97100. 
The following experimental data illustrate the utility of the fungicidal compositions of the present invention.